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Transgenic animals are organisms that have had their genome modified through the deliberate introduction of foreign DNA. This document discusses various applications of transgenic animals in biomedical research and agriculture. Transgenic mice, goats, pigs, and other animals are used as models of human diseases to study gene function and test potential treatments. The milk of transgenic goats and rabbits can be used to produce therapeutic proteins. Transgenic chickens are a promising system for producing recombinant proteins in egg whites on a large scale for pharmaceutical purposes. The creation of transgenic animals generally involves microinjection of DNA into fertilized eggs.
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- 2. Transgenic animal Mo.Alashram 2 Model for diseases can produce valuable pharmaceuticals information
- 3. Transgenic animal Mo.Alashram 3 can be defined as modification of the genetic of an organism through
- 4. Transgenic animal Mo.Alashram 4 European Laboratory Animal Associations as an animal in which there has been a deliberate modification of its , the genetic there has been a deliberate modification of its genome
- 5. Transgenic animal Mo.Alashram 5 WHY? Study gene function and regulation Genome mapping
- 6. Transgenic animal Mo.Alashram 6 • Test knock in gene Knock out gene
- 7. Transgenic animal Mo.Alashram 7 Cancer research Transposons transferred from galley fish
- 8. Transgenic animal Mo.Alashram 8 • The genes can be modified to jump wily-nilly around cells. By putting them into a batch of identify which genes or combinations of genes lead to cancer .
- 9. Transgenic animal Mo.Alashram 9 Jumping genes from jelly fish • By putting them into a batch of human or mouse cells, and watching to see which ones become cancerous
- 11. Transgenic animal Mo.Alashram 11 have been made using animals medical advances over the last century
- 12. Transgenic animal Mo.Alashram 12 Biomedical research a treatments before attempting to diagnose model of a disease to learn about it and test
- 13. Transgenic animal Mo.Alashram 13 Benefits of transgenic? Phytase enzyme poultry animal production
- 14. Transgenic animal Mo.Alashram 14 salmon • Improved gain efficiency and protein production Super-salmon
- 15. Transgenic animal Mo.Alashram 15 salmon Antifreeze protein in flounder + Chinook salmon
- 18. Transgenic animal Mo.Alashram 18 Embryonic Stem Cell Lines Isolate inner cell mass (destroys embryo) Heart muscle Kidney Liver “Special sauce” (largely unknown) Day 5-6 Blastocyst Inner cells (forms fetus) Outer cells (forms placenta) Heart repaired Culture cells
- 22. Transgenic animal Mo.Alashram 22 Nuclear Transfer Lamb 6LL3 (‘Dolly’) Donor: (adult) mammary gland cell - Finn Dorset ewe Recipient: enucleated oocyte - Scottish Blackface ewe
- 24. Transgenic animal Mo.Alashram 24 EX-Vivo Trans genesis
- 26. Transgenic animal Mo.Alashram 26 • 1981: First transgenic mouse – Insertion of hGH into a mouse (Singleton, 1999) – Production of monoclonal antibodies & anti-inflammatory agents
- 27. Transgenic animal Mo.Alashram 27 CAST, 2003 has been the most common method
- 28. Transgenic animal Mo.Alashram 28 2. An animal is given hormonal treatment to produce a large number . Of embryos, and the embryos are collected from the oviduct. 3. The human gene is inserted into the fertilized egg via microinjection 4. The transgenic embryo is placed in a surrogate host which gives Birth to the transgenic animal 5. The offspring is tested for the new gene 1. A human gene responsible for producing a desired protein Is isolated in a laboratory
- 29. Transgenic animal Mo.Alashram 29 transgenic • gene transfer is the production of transgenic dairy farm
- 30. Transgenic animal Mo.Alashram 30 PEW Initiative, 2005 FDA, 2005 Mastitis Disease Resistance Annie, born March 3, 2000, is a clone of a pure-bred Jersey calf genes for Staphylococcus aurous bacteria producing lysostaphin, a protein that kills Staphylococcus aurous bacteria
- 31. Transgenic animal Mo.Alashram 31 Lysozyme is a protein found in the tears gene for an antibacterial enzyme found in human breast milk , Lysozyme inhibits the growth of bacteria by destroying the bacterial cell wall Treatment infant diarrhea produce human lysozyme in their milk
- 32. Transgenic animal Mo.Alashram 32 Transgenic goat milk contains a protein that could be extracted to make a drug for coronary bypass patients. now in human clinical trials . Millie is a Her The protein,, is called anti-thrombin III goat. transgenic
- 33. Transgenic animal Mo.Alashram 33 Steps • the gene that produces AT III is sequenced, . build a synthetic gene and make many copies and
- 34. Transgenic animal Mo.Alashram 34 then • The synthetic gene is then attached to the gene for casein , which acts as a promoter gene . This ensemble is then injected into a . newly fertilized egg
- 35. Transgenic animal Mo.Alashram 35 then • . During the first few critical cell divisions, the gene may become attached to the goat's DNA.
- 36. Transgenic animal Mo.Alashram 36 then • If successful, the new gene is now a transgenic which will become incorporated into all cells during subsequent divisions
- 37. Transgenic animal Mo.Alashram 37 then • The embryo is then transferred to a surrogate mother
- 39. Transgenic animal Mo.Alashram 39 • Cloning through the ages: – 1952: Cloning via nuclear transfer (frog) – 1989-1990: First mammals cloned – 1995: First cloning via cultured mammalian cells – 1997: First cloning via adult cells – DOLLY
- 40. Transgenic animal Mo.Alashram 40 history • First cloning via transgenic adult cells – POLLY – 1998 – 2000: Cloning of cattle, pigs, mice, goats and monkeys using adult cells – 2001: First reported cloned human embryo – 2002: First cloned pet
- 41. Transgenic animal Mo.Alashram 41 How many genes are there E. coli 4.6 Mb 4,288 genes S. cerevisiae 13.5 Mb 6,034 genes D. melanogaster 165 Mb 12,000 genes C. elegans 97 Mb 19,099 genes H. Sapiens 3,300 Mb 40,000 genes Phenotypes
- 42. Transgenic animal Mo.Alashram 42 Which of which • Me can be genetically modified No N o No
- 44. Transgenic animal Mo.Alashram 44 Ethics • Generally scientist must be the most honest persons • They know • This is a blessing of Allah
- 45. Transgenic animal Mo.Alashram 45 In human • Research on human embryo • Is illegal • Some done it behind
- 47. Transgenic animal Mo.Alashram 47 I mean • Animal model for disease • Bioreactor for producing • Therapeutic in milk of • Goat • rabbit • Cow
- 48. Transgenic animal Mo.Alashram 48 Transgenic animals are costly: 20,000-30,000 for one animal, and low chances of success. If successful, one animal can produce in its life time 200,000- 300,000 million worth of drugs. A herd of 600 transgenic cows could supply the worldwide demand of human serum albumin (used in the treatment of burns and traumatic injuries)
- 49. Transgenic animal Mo.Alashram 49 Mention what is in your list? • Transgenic • Transgenic mouse • Gene expression regulation • Structure function • Cause and effect • In path physiological process
- 50. Transgenic animal Mo.Alashram 50 Mention what are in your list • Transgenic mouse • Transgenic rat • Transgenic rabbit • Transgenic goat • Transgenic chicken • Transgenic cow • Transgenic chicken Transgenic quail
- 51. Transgenic animal Mo.Alashram 51 Transgenic rabbit • used as model for cardiovascular diseases • Used as a model for AIDS • Used as model for cancer
- 52. Transgenic animal Mo.Alashram 52 Transgenic rabbit • The recombinant protein can be produced • low cost • Wide scale
- 53. Transgenic animal Mo.Alashram 53 What rabbit milk ? • What is can be in rabbit milk • One of the most attractive protein • Is alfa glycosidase
- 55. Transgenic animal Mo.Alashram 55 when multiple preferred analysis be performed on single sample blood chronic studies long term monitoring
- 56. Transgenic animal Mo.Alashram 56 Transgenic chicken • to understand normal and abnormal embryo development limb deformities spinal bifida avian stem cells in embryos Is the coming
- 58. Transgenic animal Mo.Alashram 58 Egg • A typical egg white contains 3.5-4.0 grams of protein
- 59. Transgenic animal Mo.Alashram 59 Egg recombinant protein could yield up to a gram or more of the protein in the naturally sterile egg
- 60. Transgenic animal Mo.Alashram 60 Protein of therapeutic significance • interferon beta-1a, in the whites of eggs laid by transgenic hens using the employs Lent Vector. Interferon-
- 63. Transgenic animal Mo.Alashram 63 lentivirus • lent viral vectors have the advantage of infecting both dividing and non- dividing cells. • However, they retain stable
- 65. Transgenic animal Mo.Alashram 65 lentivirus Lentivirus can be pseudo typed with ease to infect certain tissues and cell lines with greater efficiency. Made to accommodate expression
- 66. Transgenic animal Mo.Alashram 66 lentivirus • these viral vectors do not elicit immune responses in vivo can be concentrated to titers of 10 . 109 Cfu/ ml
- 67. Transgenic animal Mo.Alashram 67 difficulty • embryos contain about 50,000 cells before the egg is laid inserting DNA into just one cell . gene transfer in other mammals involves
- 68. Transgenic animal Mo.Alashram 68 advantages • chicken's generation time is around 6 months shorter than the generation time of a large mammal , such as a goat, which requires 18 months
- 69. Transgenic animal Mo.Alashram 69 Advantage of egg white • eggs a hen lays annually 330 eggs contains • 6.5 grams of various proteins advantage of producing pharmaceuticals in eggs is that egg Is simpler Than milk milk Egg
- 70. Transgenic animal Mo.Alashram 70 Egg white advantages methods for easily extracting various proteins from eggs.
- 71. Transgenic animal Mo.Alashram 71 transgenic Other important fields neurobiology model cardiovascular biology
- 74. Transgenic animal Mo.Alashram 74 The MOUSE Life span: approx. 2.5 years Gestation : 21 days Litter size: 8 to 12 Generation time: three months Several inbred and outbreed strains Genomic database Most advance genetic technologies Cost per mouse 5 E p Housing cost Over 90% identical to human genome Large enough for physiological studies
- 77. Transgenic animal Mo.Alashram 77 NON-TRANSGENIC TRANSGENIC No modifications to the genome. Modifications to the genome. Germ line mutations Somatic cell mutations
- 78. Transgenic animal Mo.Alashram 78 Special facilities • Specialized glassware Pulled, Holding and Transfer pipettes are used for manipulating and moving embryos around. All are handmade from stock glass tubing Injection pipettes are machine pulled. • Microscopes - Surgical, Dissecting, & Inverted • Micromanipulators – Convert gross hand movements into micro-movements • Microsurgical instruments – Used for harvest and re-implantation of embryos • Incubator – Used when culturing embryos is required
- 81. Transgenic animal Mo.Alashram 81 Microinjection Set-Up Micromanipulator Inverted Microscope Hamilton Syringe for operating Holding Pipet Foot Pedal for operating Microinjection Pipet Nitrogen Nitrogen Powered Microinjection Apparatus
- 82. Transgenic animal Mo.Alashram 82 Pipets for Pronuclear Microinjection Internal Diameter 120 m – 180 m Internal Diameter 55 m – 70 m Internal Diameter 120 m – 180 m Pulled Holding Injection Transfer Internal Diameter 1 m – 3 m Next
- 90. Transgenic animal Mo.Alashram 90 Experimental Outline Procedures begin 3 days before any embryos are harvested or injected Next
- 93. Transgenic animal Mo.Alashram 93 • Pregnant mare’s serum (PMSG) is administered intraperitoneally (I.P.) to donor females • PMSG mimics follicle stimulating hormone and induces the maturation of oocytes in the ovary Superovulation Part I Next Day -3
- 94. Transgenic animal Mo.Alashram 94 super ovulation • Super ovulate donor females to maximize embryo yield
- 95. Transgenic animal Mo.Alashram 95 Day -1 • Human Chrionic Gonadotropin (HCG) is administered I.P. to donor females • HGC mimics Latinizing hormone and stimulates the ovulation of the mature oocytes from the ovary Superovulation Part II Next
- 96. Transgenic animal Mo.Alashram 96 • Set-up mating of donor male and female mice and recipient females with vasectomized males Donor females are then placed with donor males
- 97. Transgenic animal Mo.Alashram 97 harvest • Harvest embryos from donor females
- 98. Transgenic animal Mo.Alashram 98 Embryo Harvest An abdominal incision is made and the ovaries, oviducts and uteri are excised Next
- 99. Transgenic animal Mo.Alashram 99 Ovary Fat Pad Oviduct Uterus Isolating the Oviduct The structures of the excised portion of the reproductive tract are identified and the mesentery torn away to facilitate isolation of the oviduct Mesentery Next
- 100. Transgenic animal Mo.Alashram 100 Isolating the Oviduct The oviduct, along with a small portion of the uterus is isolated and removed Next
- 101. Transgenic animal Mo.Alashram 101 Fat pad Oviduct Freeing Embryos from Oviduct Cumulus Mass Cumulus Mass The cumulus mass is visualized through the wall of the oviduct and a needle is used to tear it open and free the embryos Next
- 102. Transgenic animal Mo.Alashram 102 Media with hyaluronidase Wash and Grade Embryos The cumulus mass is placed in a large drop of medium with hyalurdonidanse. After the cumulus cells have fallen away, the embryos are transferred through three small rinse drops and graded for quality. Only embryos suitable for injection advance to the final drop. Rinse Drops Embryos for injection Next
- 103. Transgenic animal Mo.Alashram 103 Grading Embryos • Healthy, fertilized embryos must be separated from degenerate and unfertile embryos for injection Next
- 104. Transgenic animal Mo.Alashram 104 Zona Pellucida Embryo Pronuclear Polar Bodies Basic Embryo Anatomy Next
- 105. Transgenic animal Mo.Alashram 105 Examples of Embryos Good Unfertilized Cleaved Degenerate Next
- 106. Transgenic animal Mo.Alashram 106 Photograph showing examples of good and degenerate embryos Next
- 107. Transgenic animal Mo.Alashram 107 Embryos in Injection Dish Acrylic Frame Glass Cover Slip Embryo injections are performed on a special microscope slide composed of an acrylic frame with a glass cover slip attached. Embryos are placed in the center of a drop of medium that is covered with oil to prevent movement and dehydration of the drop. Next
- 108. Transgenic animal Mo.Alashram 108 grading • Characteristics of a good embryo: – 2 visible pronuclear – Embryo should fill the zone pellucida – Well defined embryo within the zona pellucida
- 110. Transgenic animal Mo.Alashram 110 implantation • Surgically re-implant injected embryos into pseudo pregnant recipients
- 111. Transgenic animal Mo.Alashram 111 Pronuclear Microinjection Using gentle suction, the embryos are held in place by the holding pipet so that the pronucleus can be injected Holding Pipet Next
- 113. Transgenic animal Mo.Alashram 113 Embryo Transfer • After all embryos have been injected they are inspected and the number of embryos that still appear healthy are separated for injection. Embryos which did not survive the injection process are discarded • Using a remote mouth pipe ting device, embryos are loaded into a transfer pipette for re-implantation into pseudopregnant recipients • 20-30 embryos will be transferred into one oviduct of each recipient
- 114. Transgenic animal Mo.Alashram 114 Loading the Transfer Pipet Mineral Oil Air Embryos in media Alternate Transfer Pipet Design The transfer pipette is filled with medium then loaded with the embryos to be re-implanted in oviduct of the recipient female
- 115. Transgenic animal Mo.Alashram 115 An incision is made in the flank of an anesthetized mouse The ovary and oviduct are then gently pulled through the incision. A tear is made in the bursa and the infundibulum exposed. The tip of the loaded transfer pipette is placed inside the infundibulum and the embryos gently blown into the oviduct The tract is returned to the abdomen and the incision site is closed. Oviduct Transfer